Molecular dynamics of hemoglobin subunits as seen by fluorescence spectroscopy.

Fluorescent conjugates of beta A subunits and their respective heme-free derivatives have been prepared in which a 1,5-N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonate probe has been specifically placed at the beta-93 or beta-112 cysteine. The fluorescence anisotropy decay and static fluorescence polarization of these conjugates have been examined. Fluorescence measurements have also been made using 1-anilino-8-naphthalenesulfonate complexes, as well as the intrinsic fluorescence of the tryptophan groups. For the cases of the beta-93 and beta-112 conjugates there is substantial evidence for internal rotational freedom of the subunits. The internal mobility of the polypeptide is especially pronounced for the beta-112 conjugate. In contrast, the 1-anilino-8-naphthalenesulfonate probe placed within the heme pocket shows no indication of any rotation, other than that associated with the entire beta-subunit. Tryptophan fluorescence has been measured for the apo-beta subunits and for the peptides beta (1-55) from hemoglobins A and S. Perrin-Weber plots show the presence of multiple rotational modes suggesting mobility of the tryptophan groups.